High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy

Abstract

Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging. To this end we have built and characterized a correlated single molecule localization microscope (SMLM)/AFM that allows localizing specific, labeled proteins within high-resolution AFM images in a biologically relevant context. Using direct stochastic optical reconstruction microscopy (dSTORM)/AFM, we directly correlate and quantify the density of localizations with the 3D topography using both imaging modalities along (F-)actin cytoskeletal filaments. In addition, using photo activated light microscopy (PALM)/AFM, we provide correlative images of bacterial cells in aqueous conditions. Moreover, we report the first correlated AFM/PALM imaging of live mammalian cells. The complementary information provided by the two techniques opens a new dimension for structural and functional nanoscale biology.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

“…In this device, the optical path was aligned with the AFM cantilever. They first confirmed the performance of the correlative microscopy via imaging polymerized actin filaments on a coverslip with both imaging modalities (AFM imaging and STORM imaging); then, they validated that the correlative microscopy can be used to image live mammalian cells, thereby providing a new method for the investigations of structure-function relationships at the single-molecule level [15]. …”

“…Additionally, the excitation spectra of fluorophores must be away from the AFM laser to avoid bleaching these fluorophores. Particularly, the imaging buffer for STORM degrades samples [15], which can limit the combination of STORM and AFM to achieve a truly synchronized correlation. Therefore, it is necessary to further develop small molecule markers and optimize the imaging buffer for both AFM and super-resolution fluorescence microscopy.…”

“…To correlate the results from both AFM and super-resolution fluorescence microscopy, it is indispensable to overlay images obtained by correlative microscopy with a precision down to 10 nm. To achieve this goal, we need to optimize procedures to align these correlative images according to the identical structure of these correlative images or fiduciary markers, such as quantum dots [15]. In a word, we need to substantially improve and enhance both AFM and super-resolution fluorescence microscopy and then integrate the two techniques to achieve a high degree of matching and synchronized co-localization measurement, which is adapted to the developing requirements of life sciences.…”

“…Recently, Nature highlighted the correlative microscopies, stating two microscopes are better than one [13]. At present, several correlative microscopy techniques, such as optical microscopy/EM and optical microscopy/AFM, have been developed and achieved gratifying results [14,15]. In this review, we will describe the main principles of AFM and optical microscopy, and summarize the progress of correlative optical microscopy/AFM techniques in biological research.…”

Optical and force nanoscopy in microbiology

“…Toward this goal, correlated AFM-fluorescence imaging has been exploited to track cell surface dynamics during cellular morphogenesis 157,158 . A recent study established a correlated SMLM and AFM imaging platform for localizing specific proteins within high-resolution AFM images 159 . Although correlative nanoscopy is still in its infancy, this approach offers promising prospects for the comprehensive analysis of the structure, dynamics and interactions of single molecules in microbial cells.…”